There is no general consensus regarding which accelerometer cut-off point (CoP) is most acceptable to estimate the time spent in moderate-to-vigorous physical activity (MVPA) in children and choice of an appropriate CoP primarily remains a subjective decision. Therefore, this study aimed to analyze the influence of CoP selection on the mean MVPA and to define the optimal thresholds of MVPA derived from different accelerometer CoPs to avoid overweight/obesity and adiposity in children aged 7 to 12 years. Three hundred six children participated. Physical activity (PA) was monitored for seven consecutive days using an ActiGraph accelerometer (model GT3X) and the intensity of PA was estimated using the five most frequently published CoPs. Body adiposity was assessed using a multi-frequency bioelectrical impedance analysis. There was found a wide range of mean levels of MVPA that ranged from 27 (Puyau CoP) to 231 min∙d–1 (Freedson 2005 CoP). A receiver operating characteristic curve analysis indicated that the optimal thresholds for counts per minute (cpm) and MVPA derived from the Puyau CoP was the most useful in classifying children according to their body mass index (BMI) and fat mass percentage (FM%). In the total sample, the optimal thresholds of the MVPA derived from the Puyau CoP were 22 and 23 min∙d–1 when the categories based on BMI and FM%, respectively, were used. The children who did not meet these optimal thresholds had a significantly increased risk of being overweight/obese (OR = 2.88, P < 0.01) and risk of having excess fat mass (OR = 2.41, P < 0.01). In conclusion, the decision of selecting among various CoPs significantly influences the optimal levels of MVPA. The Puyau CoP of 3 200 cmp seems to be the most useful for defining the optimal level of PA for pediatric obesity prevention.